Animals ; CRISPR-Cas Systems ; Endodeoxyribonucleases ; genetics ; metabolism ; Gene Editing ; methods ; Mice ; Transcription Activator-Like Effector Nucleases ; genetics ; metabolism

Genome editing is a genetic manipulation technique that can modify DNA sequences at the genome level, including insertion, knockout, replacement and point mutation of specific DNA fragments. The ultimate principle of genome editing technology relying on engineered nucleases is to generate double-stranded DNA breaks at specific locations in genome and then repair them through non-homologous end joining or homologous recombination. With the intensive study of these nucleases, genome editing technology develops rapidly. The most used nucleases include meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats associated Cas proteins. Based on introducing the development and principles of above mentioned genome editing technologies, we review the research progress of CRISPR/Cas9 system in the application fields of identification of gene function, establishment of disease model, gene therapy, immunotherapy and its prospect.
CRISPR-Cas Systems/genetics* ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics* ; Gene Editing ; Technology ; Transcription Activator-Like Effector Nucleases/metabolism*

Recent developments in genome editing technology using meganucleases demonstrate an efficient method of producing gene edited pigs. In this study, we examined the effectiveness of the transcription activator-like effector nuclease (TALEN) system in generating specific mutations on the pig genome. Specific TALEN was designed to induce a double-strand break on exon 9 of the porcine α1,3-galactosyltransferase (GGTA1) gene as it is the main cause of hyperacute rejection after xenotransplantation. Human decay-accelerating factor (hDAF) gene, which can produce a complement inhibitor to protect cells from complement attack after xenotransplantation, was also integrated into the genome simultaneously. Plasmids coding for the TALEN pair and hDAF gene were transfected into porcine cells by electroporation to disrupt the porcine GGTA1 gene and express hDAF. The transfected cells were then sorted using a biotin-labeled IB4 lectin attached to magnetic beads to obtain GGTA1 deficient cells. As a result, we established GGTA1 knockout (KO) cell lines with biallelic modification (35.0%) and GGTA1 KO cell lines expressing hDAF (13.0%). When these cells were used for somatic cell nuclear transfer, we successfully obtained live GGTA1 KO pigs expressing hDAF. Our results demonstrate that TALEN-mediated genome editing is efficient and can be successfully used to generate gene edited pigs.
Animals ; Antigens, CD55/genetics ; Cell Line ; DNA Breaks, Double-Stranded ; Exons/genetics ; Galactosyltransferases/*genetics ; Gene Editing/*veterinary ; Gene Knockout Techniques ; Humans ; Nuclear Transfer Techniques ; Swine ; Transcription Activator-Like Effector Nucleases/*genetics/*metabolism